Saturday, 10 May 2014

Mutant Fail

This song pretty much sums up the discovery I made on Friday.

Basically the Drosophila mutant I have (and have been performing various experiments with) is still being expressed, Which isn't great news when you've been assuming it is a null mutant (a mutation with no expression of functional protein).
The mutant is missing the first two exons and has lost it's original start (ATG) site but annoyingly it has chosen to pick up an alternate start site. This gets more tricky as there are two that are close together


If it has picked the first one then I should still be ok as it is out of frame has 8 nonsense amino acids and then hits a STOP codon. The problem is that the second one is in frame and would happily make the remainder of the protein that contains at least one domain that is vital to its function.
The unfortunate thing is that I have no way of telling which start site is being used without an antibody (which I don't have). If anyone knows of a way to do so - I'm all ears. The best I can come up with is to clone the mutant cDNA into a GFP vector and see what is expressed. Not a great experiment as the only thing it can confirm is whether a truncated protein is being produced,

Ultimately I need to make a genuine null mutant which is what my priority has to be. Somewhat worrying with 11 months left on my contract but I may as well get on with it.

In an attempt to see a positive in this revelation - it may at least explain the lack of a phenotype in the mutant I have been using. Maybe the real deal will have a phenotype that rewards the mutant making!

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